The following publications constitute a relevant prior art to the subject matter of the present invention:
1) Almendras, J. M., Duenas, C., Nacario, J., Sherwood, N. M., and Crim, L. W., 1987. In: "Proceedings of the Fish Breeding-Workshop". Singapore, April 7-10, 1987. PA1 2) Crim, L. W. and Glebe, B. D., 1984. Aquaculture 43:47-56. PA1 3) Crim, L. W., Glebe, B. D., and Scott, A. P., 1986. Aquaculture 56:139-149. PA1 4Crim, L. W., Sutterlin, A. M., Evans, D. M., and Weil, C., 1983. Aquaculture 35:299-307. PA1 5) Goren, A., Zohar, Y., Koch, Y., and Fridkin, M., 1987. In: "Proceedings of the 3rd Int. Symp. on Reprod. Physiol. of Fish". St. John's, Newfoundland, August 1987. PA1 6) Harvey, B., Nacario, J., Crim, L. W., Juario, J. V., and Marte, C. L., 1985. Aquaculture 47:53-59. PA1 7) Lee, C. S., Tamarus, C. S., Banno, J. E., Felley, C. D., Bocek, A., and Wyban, J. A. (1986). Aquaculture 52:199-205. PA1 8) Pankhurst, N. W., Van Der Kraak, G., and Peter, R. E., 1986a. Fish Physiol. Biochem. 1:45-54. PA1 9) Pankhurst, N. W., Van Der Kraak, G., Peter, R. E., and Breton, B., 1986b. Fish Physiol. Biochem. 1:163-170. PA1 10) Peter, R. E., Nahorniak, C. S., Sokolowska, M., Chang, J. P., Rivier, R. E., Vale, W. W., King, J. A., and Millar, R. P., 1985. Gen. Comp. Endocrinal. 58:231-242. PA1 11) Petri, W., Seidel, R., and Sandow, J., 1984. In "LHRH and Its Analogues--Basic and Clinical Aspects" (F. Labrie, A. Belonger, and A. Dupont, eds.) pp. 63-76. Excerpta Medica Press. PA1 12) Sanders, L. M., McRae, G. I., Vitale, K. M., Vickery, B. H., and Kent, J. S., 1984. In "LHRH and Its Analogues--Basic and Clinical Aspects (F. Labrie, A. Belonger and A. Dupont, eds.) pp. 53-62. Excerpta Medica Press. PA1 13) Schally, A. V., 1978. Science 202:18-28 PA1 14) Sherwood, N., Eiden, L., Brownstein, M., Spiess, J., Rivier, J., and Vale, W., 1983. Proc. Natl. Acad. Sci. USA 80::2794-2798. PA1 15) Stacey, N. E., Cook, A. F., and Peter, R. E., 1979. Gen. Comp.Endocrinol. 27:246-249. PA1 16) Weil, C. and Crim, L. W., 1983. Aquaculture 35:103-115 PA1 17) Zohar, Y. and Gordin, H., 1979. J. Fish Biol. 15:665-670. PA1 18) Zohar, Y., Breton, B., and Fostier, A., 1986. Gen. Comp. Endocrinol. 64:189-198. PA1 19) Zohar, Y., Pagelson, G., Tosky, M., Finkelman, Y., and Shmuel, E., 1987. In: "Proceedings of the 3rd Int. Symp. on Reprod. Physiol. of Fish". St. John's, Newfoundland, August 1987. PA1 20) U.S. Pat. No. 4,443,368 PA1 21) U.K. Published Patent Application No. 2152342 PA1 22) U.S. Pat. No. 4,410,514 PA1 23) Japanese Published Patent Application No. 80-40210 PA1 24) Lin, H. R., Peng. C., Van der Kraak, G., Peter, R. E., and Breton, B., 1986. Gen. Comp. Endocrinol. 64:389-395 PA1 25) Peter, R. E., Nahorniak, C. S., Chang, J. P. and Crim, C. W. 1894 Gen. Comp. Endocrinol. 55:337-346 PA1 26) Peter, R. E., Lin, H. R. and Van der Kraak, G., 1984. In PROCEEDINGS OF THE FISH BREEDING WORKSHOP, Singapore, Apr. 7-10, 1987. Aquaculture 74:1-10 PA1 27) Sherwood, N. M. and Harvey, B. 1986. Gen. Comp. Endocrinol. 61:13-19.
Marine aquaculture in general, and fish farming in particular, has been extensively developed in recent years. While there has been considerable success in achieving high yields in rearing fish, there has been only limited success in the manipulation of the reproductive cycles of the reared fish. Such manipulation is a prerequisite for the further development of fish farming into a major agricultural industry.
Many of the economically important fish do not reproduce spontaneously in captivity. This is the case with mullet (Mugil cephalus), rabbitfish (Siganus sp.), milkfish (Chanos chanos), striped bass (Morone saxatilis), sea bass (Dicentrarchus labrax), seabream (Sparus aurata), catfish (Clarias sp.) and others. In all these species the reproductive failure is located in the female: whereas vitellogenesis is completed, the stages that follow, namely oocyte maturation and ovulation, do not occur, and thus there is no spawning. Instead, vitellogenic follicles undergo rapid atresia.
In some fish species which do ovulate spontaneously in captivity, such as trout and salmon, both Atlantic and Pacific, e.g. Atlantic salmon (Salmo salar) and Pacific salmon (Onchorhynchus sp.), ovulation is not synchronized and thus egg collection is a very laborious task. Additionally, the subsequent hatching of the fingerlings is not synchronized and therefore the ability to create schools of fingerlings being all at about the same growing stage, which is necessary for economical feasible fish farming, becomes very difficult.
In fish indigenous to temperate zones, such as seabream, seabass, cyprinids and salmoneds, reproduction is seasonal, i. e. ovulation and subsequent spawning occur once or several times during a limited season. Inducing such fish to ovulate and spawn out of the natural spawning season might largely contribute to the management of fish farming. For one, out of season egg production will enable full utilization of the fish farm throughout the whole year, since only thereby will it be possible to have at any given time fish of all ages and thus be able to market adult fish year round.
In salmoneds smoltification is also seasonal and in some species occurs a year or more after hatching, resulting in S.sub.1 and S.sub.2 smolts (S.sub.1 and S.sub.2 smolts - smoltification occurs more than 1 year and 2 years after hatching, respectively). It may be induced earlier in the fish's life if the brood fish are induced to spawn out of the natural spawning season, which, if feasible, will have important economic consequences. Thus, for example, in various salmon species, e.g. the Pacific salmon and the Atlantic salmon, before season spawning might result in a high proportion of S.sub.0 and S.sub.1 smelts (S.sub.O smelts - smoltification occurs less than a year after hatching) and as a consequence a shortening of the period during which the fingerlings are stocked in fresh water hatchery facilities. This means a very significant saving in facilites, feed and manpower, as well as an earlier acceleration of growth rate (upon transfer of the fingerlings into sea water, their growth rate is accelerated), all of which will bring about a total reduction in the expenses related to the farming of the fish.
In many salmon hatcheries eggs are obtained from captured adults returning from the sea for reproduction. However, the egg yield is usually low since many adults return early, i.e. before they are ready to spawn, and die in captivity before spawning. The yield may be increased significantly if these early refuming adults were induced to ovulate earlier.
Ovulation and spawning in female fish are controlled by pituitary hormones, mainly the gonadotropins (GtH). However, the release of GtH is not spontaneous but rather induced by a gonadotropin releasing hormone (GNRH) which is secreted by the hypothalamus. The GriRH was found to be a decapeptide both in mammals (Schally, 1978) and in fish (U.S. Pat. No. 4,443,368). It has recently been found in female Sparus aurata, that the level of GtH in the pituitary gland increases as the fish approaches its natural spawning season, i.e. winter time. However, this accumulated GtH is not released into the blood, the consequence being that the oocytes undergo rapid atresia. In cases where ovulation and spawning do occur, this is always accompanied by a GtH surge in the blood (Stacey et al., 1979; Zohar et al., 1986, 1987). Such a surge of GtH and the subsequent ovulation and spawning may be induced by injection of GNRH or analogs thereof. The use of natural fish GNRH in inducing ovulation and spawning have been described in U.S. Pat. No. 4,443,368 and the use of various analogs thereof has been described in U.K. Published Patent Application No. 2152342 and in U.S. Pat. No. 4,410,514. The use of luteinizing hormone releasing hormones (LHRH) for inducing spawning in fish has been described in Japanese Published Patent Application 80-40210.
The known methods for inducing ovulation and spawning in fish in accordance with the prior art usually involve injecting a saline solution containing a non-toxic salt of GNRH or analogs thereof into the fish. However, due to a short lifetime of the GNRH in the blood, the effect of such a treatment is minimal and in many fish species ovulation and spawning cannot be induced. For example, a single injection of a GNRH analog at an amount of 5-10 82 g/kg body weight, induces spawning in Sparus aurata, but only in as little as 20 to 30% of the GnRH-treated females (see the experimental section of this specification). The same phenomenon was found also to occur in other fish in which there is a non-synchronous ovarian development in captivity, such as the sea bass Lates calcarifer (Almendras, et al., 1987).
It should be noted that in many fish carrying out successful injections, in accordance with the prior art, requires highly skilled personnel since in many cases, prior to injecting, the development stage of the oocytes has to be determined. This determination involves withdrawing some oocytes using a capillary which is inserted into the oviduct and then examining the so withdrawn oocytes under a microscope. Skilled personnel needed therefor are not available in most fish farms and thus the yield of success in inducing spawning will be lower than that obtained in the laboratory, e.g. in the case of Sparus aurata spawning will be induced in less than 20% of the females.
The short lifetime of GNRH in the blood is partially due to its rapid degradation by both specific endopeptidases and non-specific exopeptideases present in the pituitary, kidney and liver (Goren, et al., 1987). The degradation generally occurs at positions 5-6 and 9-10 of the decapeptides. Therefore, substitution of the amino acid located at position 6 (glycine) by certain D-amino acids results in GNRH analogs which are less susceptible to enzymatic degradation, thus prolonging their presence in the blood and hence their biological effectiveness (Peter et al., 1985; Zohar et al., 1987; Goren et al., 1987). However, even such analogs, when injected in biological effactive amounts, still disappear from circulation quite rapidly e.g., 30 to 60 min. in goldfish (Sherwood and Harvey, 1986) and 1 to 2 hours in the seabream (Zohar, unpublished results). Thus, using cleavage resistant analogs by itself is insufficient for obtaining a long lasting release of GtH from the pituitary.
one way to overcome this limitation and to obtain a long lasting Surge Of GtH in the blood is to use multiple injections of GNRH. or analogs thereof, but such multiple injections are excluded in most fish species due to the stress they involve. It has also been proposed to apply GNRH through the water in which fish are kept, but such an application is not economical for large scale, agricultural, applications.
Another approach in obtaining a long lasting surge of GtH in the blood is to administer GNRH to the fish in a sustained release delivery system and this has indeed been reported. The use of silastic implants (silicone rubber impregnated with the active compound) containing super-active mammalian GNRH analogs was shown to increase plasma GtH levels and accelerate spawning (Crim et al., 1986) and also to accelerate spermiation (Weil and Crim, 1983) in the Atlantic salmon. However, such an implant was found to be non-effective in many fish such as the female Walleye (Pankhurst et al., 1986a), in the male goldeye (Pankhurst, et al., 1986b) and in Sparus aurata (see the experimental section of this specification).
Cholesterol pellets (which are also a sustained release delivery system) containing different agonists of mammalian GNRH were successfully used to accelerate or induce ovulation and/or spawning in a number of fish such as rainbow trout (Crim et al., 1983), Atlantic salmon (Crim and Glebe, 1984), sea bass (Harvey et al., 1985) and milkfish (Lee et al., 1986). However, in addition to the fact that the preparation of the cholesterol pellets is time consuming and not practical on an industrial scale, it does not give rise to an effective manipulation reproduction.
Polymer based sustained release delivery systems for administering GNRH have been used in mammals in general and in humans in particular, with biodegradable polymers as the delivery vehicle (Sanders et al., 1984; Petri et al., 1984). However, the use of such polymer based sustained release systems in fish has not yet been reported.